Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells Kenyon G. Daniel a , Puja Gupta a , R. Hope Harbach b , Wayne C. Guida a,b , Q. Ping Dou a,* a Drug Discovery Program, Departments of Biochemistry & Molecular Biology and Interdisciplinary Oncology, H. Lee Moffitt Cancer Center & Research Institute, College of Medicine, University of South Florida, Tampa, FL, USA b Department of Chemistry, Eckerd College, St. Petersburg, FL, USA Received 18 June 2003; accepted 31 October 2003 Abstract Here we report that organic copper complexes can potently and selectively inhibit the chymotrypsin-like activity of the proteasome in vitro and in vivo. Several copper compounds, such as NCI-109268 and bis-8-hydroxyquinoline copper(II) [Cu(8-OHQ) 2 ], can inhibit the chymotrypsin-like activity of purified 20S proteasome. In human leukemia cells, proteasome inhibition occurs within 15 min after treatment, followed by apoptosis. Neither proteasome inhibition nor apoptosis occurs in non-transformed, immortalized human natural killer cells under the same treatment. Furthermore, proteasome inhibition and apoptosis induction were detected in prostate cancer cells treated with the ligand 8-OHQ alone following pre-treatment with copper(II) chloride. None of these events occurred in cells treated with copper(II) chloride alone, 8-OHQ alone (without growth in copper-enriched media), or nickel(II) chloride pre-treatment followed by 8- OHQ. Furthermore, we found that copper-mediated inhibition of purified 20S proteasome cannot be blocked by a reducing agent and that organic copper compounds do not generate hydrogen peroxide in the cells, suggesting that proteasome inhibition and apoptosis induction are not due to copper-mediated oxidative damage of proteins. Our results suggest that certain types of organic ligands could bind to tumor cellular copper, forming potent proteasome inhibitors and apoptosis inducers at copper concentrations found in tumor tissues. # 2003 Elsevier Inc. All rights reserved. Keywords: Copper; Anti-copper drugs; Chelator; Proteasome inhibitors; Drug discovery 1. Introduction Apoptosis is a highly conserved cellular suicide program in multicellular organisms from worms to humans [1–4]. This cellular death program serves as a means to maintain multicellular organisms by discarding damaged and unde- sirable cells [2,5]. Faulty execution of apoptosis, including either excessive cell death or insufficient cell death, is a factor in many disease states including AIDS and cancer [2]. Apoptosis features several distinct events and mor- phological changes, such as loss of the mitochondrial membrane potential, proteolytic dismantling of cellular components, DNA fragmentation, and cellular condensa- tion into apoptotic bodies that are removed by phagocytes [1,6]. As a distinct series of cellular pathways, apoptosis potentially offers unique targets for chemotherapeutic intervention. It has been suggested that cancer cells are more sensitive to several apoptosis-inducing stimuli than Biochemical Pharmacology 67 (2004) 1139–1151 0006-2952/$ – see front matter # 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2003.10.031 * Corresponding author. Present address: The Prevention Program, Department of Pathology, Barbara Ann Karmanos Cancer Institute, School of Medicine, Wayne State University, 516 HWCRC, 4100 John R Road, Detroit, MI 48201, USA. Tel.: þ1-313-966-0641; fax: þ1-313-966-7368. E-mail address: doup@karmanos.org (Q.P. Dou). Abbreviations: TM, tetrathiomolybdate; Trientine, triethylenetetra- mine; D-PA, D-penicillamine; Cu-TM, TM copper complex; Cu[trienti- ne]Cl 2 , trientine copper complex; Cu-D-PA, D-PA copper complex; 8-OHQ, 8-hydroxyquinoline hemisulfate; 8-OHQ-5-sulfonic acid, 8-hydroxyquino- line-5-sulfonic acid; 1,10-PT, 1,10-phenanthroline monohydrate; PDC, pyrrolidine dithiocarbamate; DBPTC, dibromo (1,10-phenanthroline)cop- per(II); DCPTC, dichloro (1,10-phenanthroline)copper(II); Cu[8-OHQ] 2 , 8-OHQ copper complex, molar ratio 2:1; Cu[phen]Cl 2 , 1,10-PT copper complex; 5,7-DCl, 5,7-dichloro-8-hydroxyquinoline; 5-N-8-OHQ, 5-ami- no-8-hydroxyquinoline; 8-OHQ-I-S, 8-hydroxy-7-iodo-5-quinoline-sulfo- nic acid; 8-OHI-Cu, 5-iodo-8-OHQ copper complex; 8-OHS-Cu, 8-OHQ- 5-sulfate copper complex; ()-EGCG, ()-epigallocatechin-3-gallate; NAC, N-acetyl-L-cysteine; DMF, N,N-dimethylformamide; ICP-OES, inductively coupled plasma optical emission spectroscopy; PARP, poly(- ADP-ribose) polymerase; Ub, ubiquitin.